The present invention generally relates to the field of material characterization. In particular, the invention relates to liquid chromatography techniques for rapidly characterizing and sampling polymer solutions, emulsions and dispersions, and to devices for implementing such techniques. The invention particularly relates to characterization of polymer samples in a comprehensive, directly-coupled, multi-dimensional high-performance liquid chromatography system including HPLC (High Performance Liquid Chromatography) and TREF (Temperature Rising Elution Fractionation)—like conditions in which temperature, and/or solvent gradients in any combination can be used during separation for determining a variety of properties, such as composition and molecular weight or size. The methods and devices disclosed herein are applicable, inter alia, to the rapid and multi-dimensional characterization of libraries of polymers prepared by combinatorial materials science techniques.
Currently, there is substantial research activity directed toward the discovery and optimization of polymeric materials for a wide range of applications. Although the chemistry of many polymers and polymerization reactions has been extensively studied, it is, nonetheless, rarely possible to predict a priori the physical or chemical properties a particular polymeric material will possess or the precise composition and architecture that will result from any particular synthesis scheme. Thus, characterization techniques to determine such properties are an essential part of the discovery process.
Liquid chromatography is well known in the art for characterizing a polymer sample. Liquid chromatographic techniques employ separation of one or more components of a polymer sample from other components thereof by flow through a chromatographic column, followed by detection of the separated components with a flow-through detector. Approaches for liquid chromatography can vary, however, with respect to the basis of separation and with respect to the basis of detection.
Gel permeation chromatography (GPC), a well-known form of size exclusion chromatography (SEC), is a frequently-employed chromatographic technique for polymer size determination. In GPC, the polymer sample is separated into components according to the hydrodynamic volume occupied by each component in solution. More specifically, a polymer sample is injected into a mobile phase of a liquid chromatography system and is passed through one or more chromatographic columns packed with porous beads. Molecules with relatively small hydrodynamic volumes diffuse into the pores of the beads and remain therein for longer periods, and therefore exit the column after molecules with relatively larger hydrodynamic volume. Hence, GPC can characterize one or more separated components of the polymer sample with respect to its effective hydrodynamic radius (Rh).
Another chromatographic separation approach is illustrated by U.S. Pat. No. 5,334,310 to Fréchet et al. and involves the use of a porous monolithic stationary-phase as a separation medium within the chromatographic column, combined with a mobile phase composition gradient. (See also, Petro et al, Molded Monolithic Rod of Macroporous Poly(styrene-co-divinylbenzene) as a Separation Medium for HPLC Synthetic Polymers: “On -Column” Precipitation-Redissolution Chromatography as an Alternative to Size Exclusion Chromatography of Styrene Oligomers and Polymers, Anal. Chem., 68, 315-321 (1996); and Petro et al, Immobilization of Trypsin onto “Molded” Macroporous Poly(Glycidyl Methacrylate-co-Ethylene Dimethacrylate) Rods and Use of the Conjugates as Bioreactors and for Affinity Chromatography, Biotechnology and Bioengineering, Vol. 49, pp. 355-363 (1996)). Chromatography involving the porous monolith is reportedly based on a precipitation/redissolution phenomenon that separates the polymer according to size—with the precipitated polymer molecules selectively redissolving as the solvent composition is varied. The monolith provides the surface area and permeation properties needed for proper separation.
Another chromatographic separation approach, TREF is a frequently-employed chromatographic technique for differentiating discrete polymer species from polymer blend samples as well as characterizing the components. In TREF, the polymer sample is separated into components according to the solubility of each component in a mobile phase at differing temperatures in solution. More specifically, a polymer sample is injected into a mobile phase of a liquid chromatography system and is passed through a chromatographic column at a low temperature, such as room temperature. The sample precipitates onto the column. The temperature of the column is raised, typically with an oven, and different species of the sample are eluted according to their solubility. Molecules with relatively low degree of solubility in the mobile phase remain in the column for longer periods, and therefore exit the column after molecules with a relatively low degree of solubility. Hence, TREF can characterize one or more separated components of the polymer sample with respect to its crystallinity. A TREF separation typically does not very mobile phase composition and can be very time consuming. Due to the constraints of heating and equilibrating the system, a typical separation can take up to a day for one sample.
Other separation approaches are also known in the art, including for example, normal-phase adsorption chromatography (with separation of polymer components being based on preferential adsorption between interactive functionalities of repeating units and an adsorbing stationary-phase) and reverse-phase chromatography (with separation of polymer components being based on hydrophobic interactions between a polymer and a non-polar stationary-phase). After separation, a detector can measure a property of the polymer or of a polymer component—from which one or more characterizing properties, such as molecular weight can be determined as a function of time. Specifically, a number of molecular-weight related parameters can be determined, including for example: the weight-average molecular weight (Mw), the number-average molecular weight (Mn), the molecular-weight distribution shape, and an index of the breadth of the molecular-weight distribution (Mw/Mn), known as the polydispersity index (PDI). Other characterizing properties, such as mass, particle size, composition or conversion can likewise be determined.
A variety of continuous-flow detectors have been used for measurements in liquid chromatography systems. Common flow-through detectors include optical detectors such as a differential refractive index detector (RI), an ultraviolet-visible absorbance detector (UV-VIS), or an evaporative mass detector (EMD)—sometimes referred to as an evaporative light scattering detector (ELSD). Additional detection instruments, such as a static-light-scattering detector (SLS), a dynamic-light-scattering detector (DLS), and/or a capillary-viscometric detector (C/V) are likewise known for measurement of properties of interest. Light-scattering methods, both static and dynamic, are established in several areas of polymer analysis. Static light scattering (SLS) can be used to measure Mw and the radii of gyration (Rg) of a polymer in a dilute solution of known concentration. Dynamic light scattering (DLS) measures the fluctuations in the scattering signal as a function of time to determine the diffusion constant of dissolved polymer chains or other scattering species in dilute solution or of polymer particles comprising many chains in a heterogeneous system such as dilute emulsion or latex dispersion. The hydrodynamic radius, Rh, of the chains or particles can then be calculated based on well-established models.
Presently known liquid chromatography systems are not suitable for efficiently screening larger numbers of polymer samples. Known chromatographic techniques utilizing composition gradients, such as HPLC, can typically take up to an hour or more for each sample to ensure a high degree of separation over the wide range of possible molecular weights (i.e., hydrodynamic volumes) for a sample, while chromatographic techniques utilizing temperature gradients, such as TREF can typically take up to a day or more for each sample to ensure a high degree of separation over the wide range of possible crystallinities for a sample. The known chromatographic techniques can be even longer if the sample is difficult to dissolve or if other problems arise. Also, there are no known systems for effectively utilizing composition and temperature gradients of the mobile phase over a wide range of temperatures and compositions.
Additionally, polymer samples are typically prepared for characterization manually and individually, and some characterization systems require specially-designed sample containers and/or substantial delay-times. For example, optical methods such as light-scattering protocols typically employ detector-specific cuvettes which are manually placed in a proper location in the light-scattering instrument. Such optical protocols can also require a sample to thermally equilibrate for several minutes before measurement. Moreover, because of the nature of many commercial polymers and/or polymer samples—such as their non-polarity and insolubility in water and/or alcohols, their heterogeneous nature, their lack of sequence specificity, among other aspects, the methods, systems and devices developed in connection with the biotechnological, pharmaceutical and clinical-diagnostic arts are generally not instructive for characterizing polymers. Hence, known approaches are not well suited to the rapid characterization of polymers.
Aspects of polymer characterization, such as sample preparation and polymer separation, have been individually and separately investigated. For example, Poché et al. report a system and approach for automated high-temperature dissolution of polymer samples. See Poché et al., Use of Laboratory Robotics for Gel Permeation Chromatography Sample Preparation: Automation of High-Temperature Polymer Dissolution, J. Appl. Polym. Sci., 64(8), 1613-1623 (1997). Stationary-phase media that reduce chromatographic separation times of individual polymer samples have also been reported. See, for example, Petro et al., Molded continuous poly(styrene-co-divinylbenzene) rod as a separation medium for the very fast separation of polymers; Comparison of the chromatographic properties of the monolithic rod with columns packed with porous and no-porous beads in high-performance liquid chromatography, Journal of Chromatography A, 752, 59-66 (1996); and Petro et al., Monodisperse Hydrolyzed Poly(glycidyl methacrylate-co-ethylene dimethacrylate) Beads as a Stationary Phase for Normal-Phase HPLC, Anal. Chem., 69, 3131 (1997). However, such approaches have not contemplated nor been incorporated into protocols and systems suitable for large-scale, or even moderate-scale, combinatorial chemistry research, and particularly, for combinatorial material science research directed to the characterization of polymers.
With the development of combinatorial techniques that allow for the parallel synthesis of arrays comprising a vast number of diverse industrially relevant polymeric materials, there is a need for methods and devices and systems to rapidly characterize the properties of the polymer samples that are synthesized.